The mammalian immune system provides a means for the recognition and elimination of tumor cells, other pathogenic cells, and invading foreign pathogens. While the immune system normally provides a strong line of defense, there are still many instances where cancer cells, other pathogenic cells, or infectious agents evade a host immune response and proliferate or persist with concomitant host pathogenicity. Chemotherapeutic agents and radiation therapies have been developed to eliminate replicating neoplasms. However, most, if not all, of the currently available chemotherapeutic agents and radiation therapy regimens have adverse side effects because they work not only to destroy cancer cells, but they also affect normal host cells, such as cells of the hematopoietic system. Furthermore, chemotherapeutic agents have limited efficacy in instances where host drug resistance is developed.
Foreign pathogens can also proliferate in a host by evading a competent immune response or where the host immune system has been compromised by drug therapies or by other health problems. Although many therapeutic compounds have been developed, many pathogens are or have become resistant to such therapeutics. The capacity of cancer cells and infectious organisms to develop resistance to therapeutic agents, and the adverse side effects of the currently available anticancer drugs, highlight the need for the development of new therapies specific for pathogenic cell populations with reduced host toxicity.
Researchers have developed therapeutic protocols for destroying cancer cells by targeting cytotoxic compounds specifically to such cells. These protocols utilize toxins conjugated to ligands that bind to receptors unique to or overexpressed by cancer cells in an attempt to minimize delivery of the toxin to normal cells. Using this approach certain immunotoxins have been developed consisting of antibodies directed to specific receptors on pathogenic cells, the antibodies being linked to toxins such as ricin, Pseudomonas exotoxin, Diptheria toxin, and tumor necrosis factor. These immunotoxins target tumor cells bearing the specific receptors recognized by the antibody (Olsnes, S., Immunol Today, 10, pp. 291-295, 1989; Melby, E. L., Cancer Res., 53(8), pp. 1755-1760, 1993; Better, M. D., PCT Publication Number WO 91/07418, published May 30, 1991).
Another approach for selectively targeting populations of cancer cells or foreign pathogens in a host is to enhance host immune response against the pathogenic cells, thereby avoiding the need for administration of compounds that may also exhibit independent host toxicity. One reported strategy for immunotherapy is to bind antibodies, for example, genetically engineered multimeric antibodies, to the tumor cell surface to display the constant region of the antibodies on the cell surface and thereby induce tumor cell killing by various immune-system mediated processes. (De Vita, V. T., Biologic Therapy of Cancer, 2d ed. Philadelphia, Lippincott, 1995; Soulillou, J. P., U.S. Pat. No. 5,672,486). However, this approach has been complicated by the difficulties in defining tumor-specific antigens. Another approach to relying on host immune competency is the targeting of an anti-T cell receptor antibody or anti-Fc receptor antibody to tumor cell surfaces to promote direct binding of immune cells to tumors (Kranz, D. M., U.S. Pat. No. 5,547,668). A vaccine-based approach has also been described which relies on a vaccine comprising antigens fused to cytokines, with the cytokine modifying the immunogenicity of the vaccine antigen, and, thus, stimulating the immune response to the pathogenic agent (Pillai, S., PCT Publication Number WO 91/11146, published Feb. 7, 1991). That method relies on indirect modulation of the immune response reported. Another approach for killing unwanted cell populations utilizes IL-2 or Fab fragments of anti-thymocyte globulin linked to antigens to eliminate unwanted T cells; however, based on reported experimental data, the method appears to eliminate only 50% of the targeted cell population, and results in nonspecific cell killing in vivo (i.e., 50% of peripheral blood lymphocytes that are not T cells are also killed (Pouletty, P., PCT publication number WO 97/37690, published Oct. 16, 1997)). Thus, there remains a significant need for therapies directed to treatment of disease states characterized by the existence of pathogenic cell populations in an affected host.
The present invention is directed to a method of eliminating pathogenic cell populations in a host by increasing host immune system recognition of and response to such cell populations. Effectively, the antigenicity of the cellular pathogens is increased to enhance the endogenous immune response-mediated elimination of the population of pathogenic cells. The method avoids or minimizes the use of cytotoxic or antimicrobial therapeutic agents. The method comprises administration of a ligand-immunogen conjugate wherein the ligand is capable of specific binding to a population of pathogenic cells in vivo that uniquely expresses, preferentially expresses, or overexpresses a ligand binding moiety, and the ligand conjugated immunogen is capable of eliciting antibody production or, more preferably, capable of being recognized by endogenous or co-administered exogenous antibodies in the host animal. The immune system mediated elimination of the pathogenic cells is directed by the binding of the immunogen conjugated ligand to a receptor, a transporter, or other surface-presented protein uniquely expressed, overexpressed, or preferentially expressed by the pathogenic cell. A surface-presented protein uniquely expressed, overexpressed, or preferentially expressed by the pathogenic cell is a receptor not present or present at lower amounts on non-pathogenic cells providing a means for selective elimination of the pathogenic cells. At least one additional therapeutic factor, for example, an immune system stimulant, a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, a cytotoxic immune cell, or an antimicrobial agent may be co-administered to the host animal to enhance therapeutic efficiency.
In one embodiment, the present method includes the steps of administering ligands capable of high affinity specific binding in vivo to cell surface proteins uniquely expressed, preferentially expressed, or overexpressed on the targeted pathogenic cell population, said ligands being conjugated to immunogens against which an innate or an acquired immunity already exists or can be elicited in the host animal, and optionally co-administration of at least one therapeutic factor that is an endogenous immune response activator or a cytotoxic compound. In one preferred embodiment the method involves administering a ligand-immunogen conjugate composition to the host animal wherein the ligand is folic acid or another folate receptor binding ligand. The ligand is conjugated, for example, by covalent binding, to an immunogen capable of eliciting an antibody response in the host animal or, more preferably, an immunogen capable of binding to preexisting endogenous antibodies (consequent to an innate or acquired immunity) or co-administered antibodies (i.e., via passive immunization) in the host animal. At least one additional therapeutic factor, not capable of specific binding to the ligand-immunogen complex, but capable of stimulating or enhancing an endogenous immune response, a cell killing agent, a tumor penetration enhancer, such as an inflammatory or proinflammatory agent, a chemotherapeutic agent, a cytotoxic immune cell, or an antimicrobial agent can be administered to the host animal in conjunction with administration of the ligand-immunogen conjugates.
In accordance with another embodiment of the invention there is provided a method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population wherein the members of said cell population have an accessible binding site for a ligand. The method comprises the step of administering to said host a ligand-immunogen conjugate composition comprising a complex of the ligand and an immunogen wherein said immunogen is known to be recognized by an endogenous or an exogenous antibody in the host or is known to be recognized directly by an immune cell in the host, and at least one additional composition comprising a therapeutic factor, said factor being selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response wherein the compound does not bind to the ligand-immunogen conjugate.
In accordance with an alternative embodiment of the invention, there is provided a method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population wherein said population expresses a binding site for a ligand. The method comprises the steps of administering to the host a composition comprising a complex of said ligand and an immunogen, administering to the host antibodies directed against the immunogen, and administering to said host at least one additional therapeutic factor, said factor being selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a stimulant of an endogenous immune response that does not bind to the ligand-immunogen complex.
In one preferred embodiment of the invention, there is provided a method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population wherein said population preferentially expresses, uniquely expresses, or overexpresses a folic acid receptor. The method comprises the step of administering to said host a composition comprising a covalently linked conjugate of an immunogen wherein the immunogen is known to be recognized by an endogenous or exogenous antibody in the host or is known to be recognized directly by an immune cell in the host, and a ligand comprising folic acid or a folic acid analogue having a glutamyl group wherein the covalent linkage to the immunogen is only through the γ-carboxy group of the glutamyl group. In another embodiment at least one additional composition is administered to the host comprising a therapeutic factor, said factor being selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response wherein the compound does not bind to the ligand-immunogen conjugate.
In yet another embodiment of the invention, there is provided a method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population wherein said population preferentially expresses, uniquely expresses, or overexpresses a folic acid receptor. The method comprises the step of administering to said host a composition comprising a covalently linked conjugate of an immunogen wherein the immunogen is known to be recognized by an endogenous or exogenous antibody in the host or is known to be recognized directly by an immune cell in the host, and a ligand comprising folic acid or a folic acid analogue having a glutamyl group wherein the covalent linkage to the immunogen is only through the α-carboxy group of the glutamyl group. In another embodiment at least one additional composition is administered to the host comprising a therapeutic factor, said factor being selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response wherein the compound does not bind to the ligand-immunogen conjugate.
In still one other embodiment of this invention, the targeted pathogenic cell population is a cancer cell population. In another embodiment the targeted cell population are virus-infected endogenous cells. In another embodiment the targeted cell population is a population of exogenous organisms such as bacteria, mycoplasma yeast or fungi. The ligand-immunogen conjugate binds to the surface of the tumor cells or pathogenic organisms and “labels” the cell members of the targeted cell population with the immunogen, thereby triggering an immune mediated response directed at the labeled cell population. Antibodies administered to the host in a passive immunization or antibodies existing in the host system from a preexisting innate or acquired immunity bind to the immunogen and trigger endogenous immune responses. Antibody binding to the cell-bound ligand-immunogen conjugate results in complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, antibody opsonization and phagocytosis, antibody-induced receptor clustering signaling cell death or quiescence or any other humoral or cellular immune response stimulated by antibody binding to cell-bound ligand-immunogen conjugates. In cases where an antigen can be directly recognized by immune cells without prior antibody opsonization, direct killing of pathogenic cells can occur.
Elimination of the foreign pathogens or infected or neoplastic endogenous cells can be further enhanced by administering a therapeutic factor capable of stimulating an endogenous immune response, a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, a cytotoxic immune cell, or an antimicrobial agent. In one embodiment, the cytotoxic immune cell is a cytotoxic immune cell population that is isolated, expanded ex vivo, and is then injected into a host animal. In another embodiment of the invention an immune stimulant is used and the immune stimulant may be an interleukin such as IL-2, IL-12, or IL-15 or an IFN such as IFN-α, IFN-β, or IFN-γ, or GM-CSF. In another embodiment the immune stimulant may be a cytokine composition comprising combinations of cytokines, such as IL-2, IL-12 or IL-15 in combination with IFN-α, IFN-β, or IFN-γ, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines.
In still one other embodiment of the invention, there is provided a pharmaceutical composition comprising therapeutically effective amounts of a ligandimmunogen conjugate capable of specific binding to a population of pathogenic cells in a host animal to promote specific elimination of said cells by an acquired or innate immune response, co-administered antibodies, or directly by an immune cell in the host, a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response wherein the compound does not bind to the ligand-immunogen conjugate, and a pharmaceutically acceptable carrier therefor. In one embodiment the pharmaceutical composition is in a parenteral prolonged release dosage form. In another embodiment the therapeutic factor is an immune stimulant comprising a compound selected from the group consisting of interleukins such as IL-2, IL-12, IL-15, IFNs such as IFN-α, IFN-β, or IFN-γ, and GM-CSF, or combinations thereof.